O-Methylated Rapamycin Derivatives For Alleviation And Inhibition Of Lymphoproliferative Disorders

ABSTRACT

The present invention relates to methods of alleviating and inhibiting a lymphoproliferative disorder in a mammal, the method comprising administering one or more rapamycin derivatives (including rapamycin) to the mammal. Further, the invention provides a method for identifying agents which are useful for alleviating and inhibiting a lymphoproliferative disorders, as well as a method for identifying agents which are capable of inhibiting metastasis of lymphatic tumors in a mammal.

BACKGROUND OF THE INVENTION

Post-transplant lymphoproliferative disorders (PTLDs) which usuallyinvolve expansion of B lymphocytes infected with the Epstein-Barr virus(EBV), are a life-threatening complication of the immunosupressivetherapy necessary to inhibit graft rejection (Morrison et al., 1994, Am.J. Med. 97:14-24; Warnke et al., 1995, AFIP Fascicle 14:531-535). PTLDscomprise a whole spectrum of lymphoproliferative disorders ranging froma polyclonal atypical lymphoid hyperplasia to a monoclonal, overtlymalignant B-cell lymphoma (Morrison et al., 1994, Am. J. Med. 97:14-24;Warnke et al., 1995, AFIP Fascicle 14:531-535; Curtis et al., 1999,Blood 94:2208-2216; Harris et al., 1997, Semin. Diagn. Path. 14:8-14).Less advanced forms of PTLDs respond to a less aggressive course ofimmunosuppressive therapy (Morrison et al., 1994, Am. J. Med. 97:14-24;Sigal et al., 1992, Ann. Rev. Immunol. 10:519-60). However, lowering thedose of standard immunosuppressive drugs, which nullify the body'sability to reject and destroy foreign tissue, can jeopardize thesurvival of a graft. Moreover, this modification in treatment withconventional agents is not effective against malignant, lymphoma-typePTLDs which are usually fatal for the graft recipient.

Lymphoma causes significant morbidity and mortality, accounting for morethan 50,000 new diagnoses annually in the United States alone. Manylymphomas are either Hodgkin's or non-Hodgkin's lymphomas, which can bederived from peripheral, mature B, T, or NK lymphomas. Based on theirnatural course, non-Hodgkin's lymphomas are classified into low,intermediate, and high grades. Low grade lymphomas are usually slowlyprogressive, but are essentially non-curable. The current 5-yeardisease-free, post-therapy survival rate for the intermediate and highgrade lymphomas is approximately 60%. These aggressive types of lymphomaresult in a rapid demise of the patients who do not respond to therapy.Prognosis of lymphomas occurring in patients who are immunocompromisedsuch as AIDS and post-transplant patients, is particularly poor.Therefore, new treatment modalities are needed to improve cure rate oflymphoma.

SDZ RAD (40-O-{2-hydroxyethyl}-rapamycin) is one of a class of rapamycinderivatives which exhibit immunosuppressive activities (PCT applicationWO 94/09010; Schuurman et al., 1997, Transplantation 64:32-5; Schuler etal. 1997, Transplantation 64:36-42; Sedrani, et al., 1998, Transplant.Proc. 30:2192-2194; Schuurman et al., 1998, Transplant Proc.30:2198-2199; Hausen et al., 1999, J. Heart Lung Transplant 18:150-159).Compounds of this class, including rapamycin, have several points ofaction in normal T lymphocytes. They inhibit primarily down-streamsignaling events mediated by the IL-2 receptor (Seghal, 1998, Clin.Biochem. 31:335-340) and other cytokine receptors (Sakata et al., 1999,Immunology Letters 68:301-309), but also affect cell-cycle progressionat the early G₁ phase (Terada et al., 1993, J. Cell Physiol. 154:7-15;Flanagan et al., 1993, Ann. N.Y. Acad. Sci. 696:31-37). Themulti-faceted immunosuppressive activities exhibited by SDZ RAD andother O-alkylated rapamycin derivatives compounds make these compoundsversatile immunosuppressive agents.

There is a significant need for more effective therapeutic andprophylactic methods for limiting the severity and frequency oflymphoproliferative disorders such as lymphomas and PTLDs. The presentinvention satisfies this need.

BRIEF SUMMARY OF THE INVENTION

The invention includes a method of alleviating a lymphoproliferativedisorder in a human patient. The method comprises administering to thepatient, in an amount sufficient to alleviating the disorder, arapamycin derivative having the chemical structure shown in Formula I inFIG. 8. In a preferred embodiment, the rapamycin derivative is40-O-(2-hydroxy)ethyl-rapamycin. Numerous other useful rapamycinderivatives (including rapamycin itself) are described in thisdisclosure. Lymphoproliferative disorders that can be alleviated usingthis method include, for example, PTLDs and lymphatic cancers such aslymphomas. The method can also be used to alleviate lymphoproliferativedisorders caused or associated with treatment of the patient byimmunosuppressive therapy (e.g., immunosuppressive therapy associatedwith tissue transplantation).

In another method included in the invention, a lymphoproliferativedisorder is inhibited or prevented in a patient at risk for developingsuch a disorder (e.g., an immunocompromised patient or a patientundergoing immunosuppressive therapy).

In these methods, the rapamycin derivative can be co-administered (in asingle composition or in discretely-administered compositions) with asecond pharmacologically active agent, such as an immunosuppressiveagent. Immunosuppressive agents are known for use in methods ofinhibiting graft rejection, and those known methods can be improved byadministering both the immunosuppressive agent and a rapamycinderivative disclosed herein to a patient who has received a graft.

The invention also includes a method of inhibiting metastasis of alymphatic tumor in a human patient afflicted with a lymphatic cancer.This method comprising administering to the patient, in an amountsufficient to inhibit lymphocyte proliferation, a rapamycin derivativehaving the chemical structure shown in Formula I.

In another aspect, the invention includes a method of assessing whetheran agent is useful for alleviating or inhibiting a lymphoproliferativedisorder in a human patient. This method comprising transforming a Blymphocyte with an Epstein-Barr virus, injecting the lymphocyte into amouse having a severe combined immunodeficiency, administering the agentto the mouse, and monitoring tumor growth in the mouse for at leastabout 21 days. If one or more of tumor regression, tumor eradication,and absence of a second tumor is observed in the mouse, then this is anindication that the agent is useful for alleviating or inhibiting apost-transplant lymphoproliferative disorder in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which illustrates SDZ RAD-mediated inhibition of invitro proliferation of PTLD-like EBV+B cells. BC-1 is an EBV+/HSV8+B-cell line derived from primary effusion lymphoma (PEL). The other celllines are in vitro EBV-transformed B-cell lines derived from one of apatient with low-grade B-cell lymphoma (15A or 20A), a patient withT-cell lymphoma (LCL) or a healthy individual (A1 or A2D6). TheHTLV-I+malignant T-cell lines, HUT-102, C10MJ, and ATL-2, were includedas control cell lines.

FIG. 2 is a graph which illustrates SDZ RAD-mediated inhibition of cellcycle progression in PTLD-like B cells. Four EBV+B-cell lines werecultured for 48 hours in the presence of 0-10 nanomolar SDZ RAD, labeledwith propidium iodine, and analyzed by flow cytometry.

FIG. 3 is a graph which illustrates SDZ RAD-mediated increase inapoptotic rate of PTLD-like B cells. Three EBV+B-cell lines werecultured for 24 hours in the presence of 0-10 nanomolar SDZ RAD, labeledwith propidium iodine and anti-Annexin V antibody, and analyzed by flowcytometry.

FIG. 4, comprising FIGS. 4A-4D, is a group of images of photomicrographswhich depict the morphology and phenotype of 20A tumors xenotransplantedinto SCID mouse. In FIG. 4A, hematoxylin-eosin stain shows large celllymphoma with high mitotic rate. In FIG. 4B, immunoperoxidase stainusing an antibody which binds specifically with human CD20 (i.e., aB-cell antigen) shows cell-membrane staining in all lymphoma cells. InFIG. 4C, immunoperoxidase stain using an antibody which bindsspecifically with Ki67 (i.e., a cell proliferation-related antigen)shows nuclear staining in 50-80% of cells. In FIG. 4D, in-situhybridization for EBV-encoded EBER-1 RNA shows nuclear positivity in alllymphoma cells.

FIG. 5, comprising FIGS. 5A-5C, is a trio of graphs which illustratesSDZ RAD-mediated inhibition of in vivo growth of PTLD-like B-cells;treatment of established tumors. Fragments of tumors derived fromEBV+B-cell lines 15A (FIG. 5C), 20A (FIG. 5B), and A2 D6 (FIG. 5A) wereimplanted into recipient SCID mice. The number of mice per group isindicated in parentheses. Treatment with 5 milligrams per kilogram bodyweight per day of the drug was started when the tumors reached 0.4-0.5cm in diameter.)

FIG. 6, comprising FIGS. 6A-6C, is a trio of graphs which illustratesSDZ RAD-mediated inhibition of in vivo growth of a malignant PTLD-likeB-cells (i.e., inhibition of tumor growth). Fragments of tumors derivedfrom EBV+B-cell lines 15A (FIG. 6C), 20A (FIG. 6B), and A2D6 (FIG. 6A)were implanted into recipient SCID mice. The number of mice per group isindicated in parentheses. The treatment with 5 milligrams per kilogrambody weight per day of the drug was started 3 days prior to the tumorimplantation.

FIG. 7 depicts the chemical structure of SDZ RAD.

FIG. 8 depicts the chemical formula for O-methylated rapamycinderivatives which can be used in the methods described herein (i.e.,Formula I).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of alleviating or inhibitinglymphoproliferative disorders such as lymphomas and post-transplantlymphoproliferative disorders (PTLDs) in a mammal such as a human. Themethod comprises administering to the mammal an O-alkylated rapamycinderivative such as SDZ RAD in amount sufficient to inhibit proliferationof lymphocytes in the mammal.

O-alkylated rapamycin derivatives are known to inhibit graft rejection.Thus, when the rapamycin derivative is administered to a mammal whichhas undergone a tissue or organ graft (i.e., a xenograft),administration of one of these derivatives can both inhibit graftrejection and alleviate or prevent a PTLD. For treatment or inhibitionof a PTLD, the amount administered can be sufficient to inhibit bothgraft rejection and proliferation of lymphocytes.

Not previously recognized by others is the fact that O-alkylatedrapamycin derivatives such as those described herein can be used toalleviate or prevent lymphoma. O-alkylated rapamycin derivative such asSDZ RAD can also be used to inhibit proliferation of lymphocytes, asoccurs in a variety of lymphoproliferative disorders other than lymphoma(e.g., chronic lymphocytic leukemia). O-alkylated rapamycin derivativessuch as SDZ RAD can be used to induce in a cell, either in vitro or invivo, cell cycle arrest or apoptosis, and to inhibit lymphocyte growthand proliferation.

The O-alkylated rapamycin derivatives that are useful in the methodsdescribed herein have been described by others, as has methods formaking them. In this regard, the PCT patent application havinginternational publication number WO 94/09010 is incorporated herein byreference.

O-alkylated rapamycin derivatives that are useful in the methodsdescribed in this disclosure include those having the chemical structureshown in Formula I in FIG. 8, wherein

X is (H, H) or O;

Y is (H, OH) or O;

each of R¹ and R² is independently selected from the group consisting of—H, alkyl, thioalkyl, arylalkyl, hydroxyalkyl, dihydroxyalkyl,hydroxyalkylarylalkyl, dihydroxyalkylarylalkyl, alkoxyalkyl,acyloxyalkyl, aminoalkyl, alkylaminoalkyl, alkoxycarbonylaminoalkyl,acylaminoalkyl, arylsulfonamidoalkyl, allyl, dihydroxyalkylallyl,dioxolanylallyl, carbalkoxyalkyl, and (R³)₃ Si;

each R³ is independently selected from the group consisting of —H,methyl, ethyl, isopropyl, tert-butyl, and phenyl; and

either R⁴ is methyl or R⁴ and R¹ together form a C₂₋₅ alkylene moiety.

In the descriptions of R¹-R⁴, “alk-” or “alkyl” refers to a C₁₋₆ alkylmoiety, the moiety being branched or linear, and preferably being a C₁₋₃alkyl moiety, in which the carbon chain can, optionally, be interruptedby an ether (—O—) linkage (e.g., —CH₂—CH₂—O—CH₂—). In the descriptionsof R¹-R⁴, “ar-” or “aryl” refers to a C₅₋₈ aryl moiety, the aryl moietyoptionally having one or two nitrogen atoms in place of a carbon atom.In the descriptions of R¹-R⁴, “allyl” means —CH₂—CH═CH₂. In thedescriptions of R¹-R⁴, “acyl” refers to a C₁₋₆ alkanoyl moiety (i.e., analkyl moiety having a carbonyl {i.e., —CO—} moiety in place of amethylene moiety {i.e., —CH₂—} moiety of the alkyl moiety). In thedescriptions of R¹-R⁴, “alkylene” refers to a C₁₋₆ alkylene moiety, themoiety being branched or linear.

Examples of O-alkylated rapamycin derivatives that are suitable for usein the methods described in this disclosure include)

-   -   40-O-benzyl-rapamycin,    -   40-O-(4′-hydroxymethyl)benzyl-rapamycin,    -   40-O-[4′-(1,2 dihydroxyethyl)]benzyl-rapamycin,    -   40-O-Allyl-rapamycin,    -   40-O-[3′(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2′-en-1′-yl]-rapamycin,    -   (2′E, 4′S)-40-O-(4′,5′-dihydroxypent-2′-en-1′-yl)-rapamycin,    -   40-O-(2-hydroxy)ethoxycarbonylmethyl-rapamycin,    -   40-O-(2-hydroxy)ethyl-rapamycin,    -   40-O-(3-hydroxy)propyl-rapamycin,    -   40-O-(6-hydroxy)hexyl-rapamycin,    -   40-O-[2-(2hydroxy)ethoxy]ethyl-rapamycin,    -   40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin,    -   40-O-[(2S)-2,3-dihydroxyprop-1-yl]-rapamycin,    -   40-O-(2-acetoxy)ethyl-rapamycin,    -   40-O-(2-nicotinoyloxy)ethyl-rapamycin,    -   40-O-[2-(N-morpholino)acetoxy]ethyl-rapamycin,    -   40-O-(2-N-imidazolylacetoxy)ethyl-rapamycin,    -   40-O-[2-(N-methyl-N-piperazinyl)acetoxy]ethyl-rapamycin,    -   39-O-desmethyl-39,40-O,O-ethylene-rapamycin,    -   (26R)-26-dihydro-40-O-(2-hydroxy)ethyl-rapamycin,    -   28-O-methyl-rapamycin,    -   40-O-(2-aminoethyl)-rapamycin,    -   40-O-(2-acetaminoethyl)-rapamycin,    -   40-O-(2-nicotinamidoethyl)-rapamycin,    -   40-O-(2-(N-methyl-imidazo-2′-ylcarbethoxamido)ethyl)-rapamycin,    -   40-O-(2-ethoxycarbonylaminoethyl)-rapamycin,    -   40-O-(2-tolylsulfonamidoetlyl)-rapamycin, and    -   40-O-[2-(4′5′-dicarboethoxy-1′,2′,3′-triazol-r-yl)-ethyl]-rapamycin.

Preferred compounds for use in the methods described herein are40-O-substituted rapamycins where X and Y are both O, R² is H, R⁴ ismethyl, and R¹ is selected from hydroxyalkyl, hydroxyalkoxyalkyl,acylaminoalkyl, and aminoalkyl. Examples of preferred compounds include40-O-(2-hydroxy)ethyl-rapamycin (i.e., SDZ RAD, the structure of whichis shown in FIG. 7), 40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, and40-O-(2-acetaminoethyl)-rapamycin. Rapamycin itself (i.e., the compoundhaving the structure of Formula I, wherein R¹ and R² are each —H, R⁴ ismethyl, and each of X and Y are 0) can also be used in the methodsdescribed herein.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

A process in a cell, such as cell growth, cell cycle progression,proliferation, or tumorigenesis, is “inhibited” by a composition ormethod of treatment if, upon administering the composition to the cellor employing the method of treatment on the cell, the process is alteredrelative to the same process in a cell to which the composition was notadministered or on which the method of treatment was not employed. Forexample, if the level of proliferation in a cell is decreased followingadministration of a composition comprising SDZ RAD, as compared with thelevel of proliferation in a cell to which the composition is notadministered, then the composition inhibits proliferation in the cell.

A process in a mammal, such as tumor growth, establishment of a tumor, alymphoproliferative response, or graft rejection, is “inhibited” by acomposition or method of treatment if, upon administering thecomposition to the mammal or employing the method of treatment on themammal, the process is decreased in rate or magnitude relative to thesame process in a mammal to which the composition was not administeredor on which the method of treatment was not employed. For example, ifthe level of proliferation of lymphocytes in a mammal is decreasedfollowing administration of a composition comprising SDZ RAD, ascompared with the level of proliferation of lymphocytes in a mammal towhich the composition is not administered, then the composition inhibitsa proliferation of lymphocytes in the mammal.

A process in a cell, such as apoptosis or cell cycle arrest, is“induced” by a composition or method of treatment if, upon administeringthe composition to the cell or employing the method of treatment on thecell, the level of the process in the cell is increased relative to theprocess in a cell to which the composition was not administered or onwhich the method of treatment was not employed. For example, if thedegree, level, or likelihood of apoptosis in a cell is increasedfollowing administration of a composition comprising SDZ RAD, ascompared with the degree, level, or likelihood of apoptosis in a cell towhich the composition is not administered, then the composition inducesapoptosis in the cell.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which an O-alkylated rapamycin derivative canbe combined and which, following the combination, can be used toadminister the rapamycin derivative to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of an O-alkylated rapamycin derivative whichis compatible with any other ingredients of the pharmaceuticalcomposition and which is not deleterious to the subject to which thecomposition is to be administered.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration caninclude, but is not limited to, subcutaneous, intraperitoneal,intramuscular, infrasternal injection, and kidney dialytic infusiontechniques.

A first pharmacological agent and a second pharmacological agent are“co-administered” if the two agents are administered sufficientlyclosely in time) that the period during which the pharmacological effectattributable to the first agent is at least half its maximal valueoverlaps with the period during which the pharmacological effectattributable to the second agent is at least half its maximal value.Co-administered agents can be administered in a single compositioncontaining the agents, or they can be administered in discretecompositions.

As used herein, the term “induction of apoptosis” means a process whichcauses a cell to begin or accelerate the process of programmed celldeath.

A disorder is “alleviated” if the severity of the disorder or one of itssymptoms or the frequency with which the disorder or one of its symptomsis experienced by a patient is reduced.

A disorder is “inhibited” by a treatment if the onset of the disorder orone of its symptoms is delayed or prevented, relative to its onset inthe absence of the treatment.

DESCRIPTION

The present invention relates to a method of using an O-alkylatedrapamycin derivative to alleviate or inhibit a lymphoproliferativedisorder in a mammal. Lymphoproliferative disorders which can bealleviated or inhibited using the method described herein includepost-transplant lymphoproliferative disorders (PTLDs), lymphomas, andother disorders. This method comprises administering to the mammal asufficient amount of an O-alkylated rapamycin derivative to inhibit thedisorder. In patients who are afflicted with both a lymphoproliferativedisorder and graft rejection, an amount of the derivative sufficient toalleviate or inhibit both the disorder and the graft rejection can beadministered. For example, both rejection of a graft (i.e., atransplanted tissue or organ) and a PTLD can be alleviatedsimultaneously.

PTLDs include a wide spectrum of lymphoproliferative disorders, rangingfrom a polyclonal atypical lymphoid hyperplasia to a monoclonal, overtlymalignant B cell lymphoma. Examples of such disorders have beendescribed in the art (e.g., Morrison et al., 1994, Am. J. Med. 97:14-24;Warnke et al., 1995, AFIP Fascicle 14:531-535; Curtis et al., 1999,Blood 94:2208-2216; Harris et al., 1997, Semin. Diagn. Path. 14:8-14)and can be identified by the skilled artisan.

One class of lymphoproliferative disorders for which the methodsdescribed herein are useful are those disorders which are characterizedby infection of lymphocytes by Epstein-Barr virus (EBV). Proliferationof EBV-infected lymphocytes is a frequent complication ofimmunosuppressive therapy, and thus sometimes occurs duringimmunosupression associated with prevention of graft rejection, withcancer-related chemotherapy, or with other immune-suppressing medicalinterventions. Furthermore, proliferation of EBV-infected lymphocytescan be accelerated in immunocompromised patients, such as patientsafflicted with AIDS or other immune disorders. The O-alkylated rapamycinderivatives described herein can be used before, after, or duringtreatment of one of these other immune system-affecting disorders ortreatments. For example, a patient who is to undergo immunosuppressivetherapy can first (i.e., minutes, hours, days, or weeks beforehand) beadministered one or more O-alkylated rapamycin derivatives (includingrapamycin) in order to inhibit or prevent proliferation of lymphocytesduring the immunosuppressive therapy. One or more O-alkylated rapamycinderivatives (including rapamycin) can be co-administered (i.e., in asingle composition or in discrete compositions administered closely intime or alternately) during the course of immunosuppressive therapy inorder to decrease or prevent proliferation of lymphocytes during thetherapy. The derivative(s) need not be the same one(s), if any,administered prior to the therapy. Similarly, one or more O-alkylatedrapamycin derivatives (including rapamycin) can also (or instead) beadministered to a patient who has undergone immunosuppressive therapy,in order to alleviate, inhibit, or prevent post-therapy lymphocyteproliferation and symptoms associated therewith.

A particular method included within the scope of the invention comprisesadministering SDZ RAD to a human prior to transplantation of a tissue(i.e., an allograft or a xenograft) into or onto the body of the human.Such administration can occur before, during, or after transplantation,or at any combination of these. By way of example, SDZ RAD can beadministered to a human patient prior to, during, and after receiving anallogeneic kidney transplant. Other examples of transplants includeallogeneic heart, kidney, and liver transplants, heart valves, vasculargrafts, skin grafts, dura mater grafts, pericardium grafts, cartilagegrafts and implants, and xenogeneic transplants.

Another type of lymphoproliferative disorder that can be, but often isnot, associated with tissue transplantation is lymphatic cancers,including lymphomas and lymphocytic leukemias. The lymphocytic leukemiasinclude disorders such as acute and chronic lymphocytic leukemias.Lymphomas encompass a wide variety of cancers characterized bylymphocyte proliferation. Examples of lymphomas include AIDS-relatedlymphomas, Hodgkin's lymphoma (sometimes designated Hodgkin's disease),non-Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large cell lymphoma,T-cell lymphoma, and cutaneous T-cell lymphoma.

Regardless of whether a patient is diagnosed with a disease or exhibitssymptoms of a disease, it can be desirable to inhibit lymphocyteproliferation to a patient who harbors lymphocytes infected with EBV,particularly if the patient is expected to undergo immunosuppressivetherapy or if the patient is at particular risk for developing animmune-compromising disorder. Such patients can be identified byisolating lymphocytes from the patient and assessing the presence orabsence of all or part of the EBV genome in those lymphocytes. If EBV isdetected, administration of rapamycin or an O-alkylated rapamycinderivative can be indicated.

The methods described herein for inhibiting or alleviating alymphoproliferative disorder comprise administering rapamycin or anO-alkylated rapamycin derivative to a mammal. The precise form in whichthe compound is administered is not critical, numerous pharmaceuticalcompositions, dosage forms, and pharmaceutically acceptable carriers andexcipients being known in the art. A formulation administered to amammal can contain the rapamycin derivative as the sole active agent, orit can be admixed with one or more other active agents (e.g., animmunosuppressive agent such as azathioprine, a mycophenolic acid suchas mycophenolate mofetil, Rh_(o)(D) immune globulin, cyclosporin,tacrolimus, cisplatin, a cyclophosphamide, and leflunomide).

The invention encompasses the preparation and use of medicaments andpharmaceutical compositions comprising one or more O-alkylated rapamycinderivatives (including rapamycin) as an active ingredient. Such apharmaceutical composition may consist of the active ingredient alone,in a form suitable for administration to a subject, or thepharmaceutical composition may comprise the active ingredient and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. Administration of one ofthese pharmaceutical compositions to a subject is useful for inhibitingproliferation of lymphocytes in the subject, as described elsewhere inthe present disclosure. The active ingredient may be present in thepharmaceutical composition in the form of a physiologically acceptableester or salt, such as in combination with a physiologically acceptablecation or anion, as is well known in the art.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to other mammals. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, and mammals includingcommercially relevant mammals such as cattle, pigs, horses, sheep, cats,and dogs.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, or another route of administration, althoughparental administration is contemplated to be the most readily effectivemethod of administering rapamycin derivatives for the purpose ofalleviating, inhibiting, or preventing lymphoproliferative disorders.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient. A unit dose of a pharmaceutical composition of the inventionwill generally comprise from about 50 micrograms to about 50 milligramsof the active ingredient, and preferably comprises from about 500micrograms to about 10 milligrams of the active ingredient. In general,O-alkylated rapamycin derivatives can be used in amounts on the sameorder of magnitude as presently-used amounts of rapamycin (e.g., 1-5milligrams per unit dose).

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include immunosuppressive agents such as azathioprine, amycophenolic acid such as mycophenolate mofetil, Rh_(o)(D) immuneglobulin, cyclosporin, tacrolimus, cisplatin, a cyclophosphamide, andleflunomide.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules, in multi-dose containerscontaining a preservative, or in single-use devices for auto-injectionor injection by a medical practitioner. Formulations for parenteraladministration include, but are not limited to, suspensions, solutions,emulsions in oily or aqueous vehicles, pastes, and implantablesustained-release or biodegradable formulations. Such formulations mayfurther comprise one or more additional ingredients including, but notlimited to, suspending, stabilizing, or dispersing agents. In oneembodiment of a formulation for parenteral administration, the activeingredient is provided in dry (i.e. powder or granular) form forreconstitution with a suitable vehicle (e.g. sterile pyrogen-free water)prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution may be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

A pharmaceutical composition of the invention may be administered todeliver a dose of between 10 micrograms per kilogram body weight per dayand 1.5 milligrams per kilogram body weight per day and, and preferablyto deliver of between 20 and 700 micrograms per kilogram body weight perday, to a subject. For adult humans, administration of 0.1 to 50milligrams per day of rapamycin or an O-alkylated rapamycin derivative,and preferably 1 to 10 milligrams per day of the active ingredient iscontemplated.

It is understood that the ordinarily skilled physician or veterinarianwill readily determine and prescribe an effective amount of the compoundto alleviate or inhibit a lymphoproliferative disorder in the subject.In so proceeding, the physician or veterinarian may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. It is furtherunderstood, however, that the specific dose level for any particularsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and theseverity of the lymphoproliferative disorder being treated or inhibited.

Another aspect of the invention relates to a kit comprising apharmaceutical composition of the invention and an instructionalmaterial. As used herein, an “instructional material” includes apublication, a recording, a diagram, or any other medium of expressionwhich is used to communicate the usefulness of the pharmaceuticalcomposition of the invention for inhibiting proliferation of lymphocytesor alleviating or inhibiting a lymphoproliferative disorder. Theinstructional material can also, for example, describe an appropriatedose of the pharmaceutical composition of the invention. Theinstructional material of the kit of the invention may, for example, beaffixed to a container which contains a pharmaceutical composition ofthe invention or be shipped together with a container which contains thepharmaceutical composition. Alternatively, the instructional materialcan be shipped separately from the container with the intention that theinstructional material and the pharmaceutical composition be usedcooperatively by the recipient.

In another embodiment, the invention includes a method of using anO-alkylated rapamycin derivative such as SDZ RAD or rapamycin to inhibitthe establishment of a tumor in a mammal. This method involvesadministering to a mammal having a first tumor, an amount of therapamycin derivative sufficient to inhibit establishment of a secondtumor derived from the first tumor in the mammal. A mammal to be treatedwith an O-alkylated rapamycin derivative for this purpose can havesubstantially any type of first tumor such as a tumor of the brain,kidney, liver, breast, or ovary. Other representative anti-tumoractivities that can be exhibited by an O-alkylated rapamycin derivativesuch as SDZ RAD include inhibition of tumorigenesis, inhibition ofmetastasis, inhibition of tumor cell growth, inhibition of tumor cellproliferation, and induction of tumor cell death.

In alternative embodiments, the present invention relates to methods ofusing an O-alkylated rapamycin derivative such as SDZ RAD to affectbiological) processes and elicit therapeutic responses related to theanti-proliferative, anti-tumor, and immunosuppressive activitiesexhibited by this compound. These methods include, for example, usingthe derivative to induce apoptosis, inhibit cell proliferation, or foranti-tumor, anti-PTLD, or immunosuppressive purposes. Thus, thederivative can be used to inhibit (i.e., ameliorate, prevent, or reducethe severity, frequency, rate, or extent) disorders associated withthese processes. As effective anti-proliferative agents, O-alkylatedrapamycin derivatives can be useful for alleviating substantially anydisorder that is characterized by supra-normal or otherwiseinappropriate cellular proliferation. Examples of disorders includeauto-immune disorders, allergies, other hyper-immune disorders,atherosclerosis.

The invention includes a method of identifying an agent that is usefulfor alleviating or inhibiting a lymphoproliferative disorder in amammal. This method comprise transforming a B lymphocyte by exposing itto EBV. Such exposure can be performed using standard reagents andtechniques and employing any available strains of EBV which cantransform lymphocytes. B lymphocytes useful for this method can, forexample, be obtained from a human donor. Transformed B lymphocytes areinjected into a mouse having a severe combined immunodeficiency, such asa SCID mouse. Transformed lymphocyte can be injected into the mouseintraperitoneally or subcutaneously. Preferably, the injection of thetransformed B lymphocyte is subcutaneous. An agent potentiallyexhibiting anti-lymphoproliferative activity is also administered to themouse. Tumor growth in the mouse is monitored for a selected period oftime, such as for about 21 days. Inhibition of tumor growth ordevelopment is an indication that the agent exhibitsanti-lymphoproliferative activity.

In one embodiment of the method, a potential anti-lymphoproliferativeagent is administered to a SCID mouse prior to the injection of atransformed B lymphocyte. By way of example, an agent such as SDZ RADcan be administered to a SCID mouse at a dose of 5 milligrams perkilogram per day for at least about 10 days, and preferably, at leastabout 3 days prior to injecting the mouse with transformed Blymphocytes.

In another embodiment of the method, a potentialanti-lymphoproliferative agent is administered to a SCID mouse after theinjection of a transformed B lymphocyte and development of the resultingtumor in the mouse. In this embodiment the injection of transformed Blymphocytes is followed by at least about 21 days of monitoring thegrowth of the resulting tumor in the mouse, and administration of apotential anti-lymphoproliferative agent to the mouse beginning aboutafter day 21. By way of example, an agent such as SDZ RAD can beadministered to a SCID mouse at a dose of 5 milligrams per kilogram perday beginning at about day 21 post-injection.

Using the methods described herein, assessment of an agent as havinganti-lymphoproliferative activity can be based on evidence of tumorregression (i.e., a decrease in tumor size relative to the initiation oftreatment with the agent), tumor eradication, and the absence of asecond tumor in the injected mouse. Likewise, an agent does not havesubstantial anti-lymphoproliferative activity if the injected mouseexhibits tumor growth (i.e., an increase in tumor size) or thedevelopment of a second tumor or both.

In other embodiments, the method described herein for identifying ananti-lymphoproliferative agent is also useful for identifying an agentcapable of inhibiting establishment of a tumor (e.g., capable ofinhibiting spread or metastasis of a lymphatic tumor) in a mammal. Forexample, an agent which inhibits establishment of a tumor in a mammalcan be identified by exposing a B lymphocyte to EPV, such that atransformed B lymphocyte is generated, administering the agent to amouse having a severe combined immunodeficiency, such as a SCID mouse,injecting the transformed B lymphocyte into the mouse, and monitoringtumor growth in the mouse for a selected period (e.g., at least about 21days). An agent capable of inhibiting establishment of a tumor willcause one or more of tumor regression, tumor eradication, and absence ofa second tumor in the injected mouse. An agent that is not capable ofinhibiting establishment of a tumor will not have an observable affecton the injected mouse in terms of tumor growth and development ofsecondary tumors.

In reference to the methods described above, procedures for measuringtumor growth or regression, injecting B lymphocytes, and determiningappropriate dosages at which to test potential anti-PTLD agents arewithin the ability of one skilled in the art of immunopharmacology. Uponreading the present disclosure and examining the particulars of Example1 provided herein, it is a simple matter for the skilled artisan to usethe methods of the present invention to identify compounds which exhibitanti-lymphoproliferative activity or which are capable of inhibitingestablishment of a tumor.

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only, andthe invention is not limited to these Examples, but rather encompassesall variations which are evident as a result of the teaching providedherein.

Example 1 SDZ RAD Inhibits Growth of Human EBV-Transformed B Lymphocytesin Vitro and in Vivo

The experiments presented in this Example demonstrate that SDZ RAD is apotent anti-rejection, anti-lymphoproliferative agent, and represents anovel approach to inhibition and treatment of post-transplantlymphoproliferative disorders (PTLDs).

The materials and methods used in the experiments presented in thisExample are now described.

Most cell lines used in this study were lymphoblastoid B-cell linesobtained by in vitro infection with EBV of peripheral blood mononuclearcells (PBMC). Cell lines A1 and A2D6 were obtained from normal, healthyindividuals. Cell lines 15A and 20A were obtained from two differentpatients with a low grade B-cell lymphoma with monoclonal coldagglutinins (Silberstein et al., 1991, Blood 78:2372-2386). Both linessecreted cold agglutinins with the same specificity as the coldagglutinins found in the patient's serum (Silberstein et al., 1991,Blood 78:2372-2386). Furthermore, the 20A cell line exhibitedcytogenetic abnormalities seen in low-grade lymphomas (48,XX, +3, +12,according to Silberstein et al., 1991, Blood 78:2372-2386). LCL B-cellline was obtained from a patient with a progressive cutaneous T-celllymphoproliferative disorder (Zhang et al., 1996, Proc. Natl. Acad. Sci.USA 93:9148-9153). BC-1 is derived from a peritoneal effision B-celllymphoma (PEL) and, in addition to EBV, harbors HHSV8 virus (Cesarman etal., 1995, Blood 86:2708-2714). The other 3 cell lines used as controlsin the in vitro growth inhibition assay were HTLV-I (+) T-cell linesATL-2 (Maeda et al., 1985, Exp. Med. 162:2169-2174); C10 MJ2 (Popovic etal., 1983, Science 219: 856-859); and HUT102B (Poiescz, 1980, Proc.Natl. Acad. Sci. USA 77:7415-7419; Bunn, et al, 1996, J. Cell. Biochem.Suppl 24:12-23) derived from patients with adult T-cellleukemia/lymphoma (ATLL). They were shown by us to be non-responsive toSDZ RAD and the related, immunosuppressive macrolide, rapamycin (Zhanget al., 1999, Leukemia Res. 23:373-384). All cell lines were maintainedin humidified incubators at 37° C. in an atmosphere containing 5% CO₂ instandard medium: RPMI Medium 1640 (Gibco BRL, Grand Island, N.Y.)supplemented with 10% (v/v) heat inactivated fetal bovine serum(BioWhittaker, Walkersville, Md.), 1% penicillin/streptomycin/fungizonemixture (Gibco BRL), and 2 millimolar L-glutamine (Gibco BRL).

An assay to assess inhibition of in vitro cell growth by SDZ RAD wasperformed as described (Zhang et al., 1999, Leukemia Res. 23:373-384;Zhang et al., 1996, Proc. Natl. Acad. Sci. USA 93:9148-9153). Briefly,cell lines were cultured for 32 hours in triplicate at 2×10⁴ cells perwell in the presence of various concentrations of SDZ RAD (NovartisPharma, Basel). After pulsing the cells with 0.5 microcurie ³H-thymidine(New England Nuclear, Boston, Mass.) and culturing the cells for thenext 18 hours, isotope incorporation into the cells was measured. Theresults of proliferation assays were expressed as the mean radioactivityof triplicate cultures. Standard deviation within the triplicates was<15%. Detection of cell cycle inhibition and apoptosis.

The cell lines to be examined were cultured with several concentrationsof SDZ RAD (0 to 10 nanomolar) for 24-48 hours. The cells were washedwith DPBS and stain solution (pH 7.2) containing 3% PEG 6000, 50micrograms per milliliter DNA fluorochrome propidium iodide (PI;Calbiochem, San Diego, Calif.), annexin V-FITC as described (Douglas etal., 1998, Cytometry 32:57-65) 0.1% (v/v) TRITON™ X (Sigma, St. Louis,Mo.), 4 millimolar citrate buffer pH 7.8, and 360 units per milliliterRNase A (Worthington Biochemicals, Freehold, N.J.) for 30 minutes at37C. Next, salt solution pH 7.2 (3% w/v PEG 6000, PI 50 micrograms permilliliter, 0.1% v/v TRITON™ X, and 0.4 molar NaCl) was added, and thecells were incubated at 4° C. in the dark for 1 hour before flowcytometry analysis was performed, as described (Douglas et al., 1998,Cytometry 32:57-65; Kawamata et al., 1998, Blood 91:561-569; Pepper etal., 1998, Leuk. Res. 22:439-444).

Five- to seven-week-old immunodeficient SCID mice (C.B-17 and ICR) werepurchased from Taconic (Germantown, N.J.) and housed at the Universityof Pennsylvania Animal Facility under pathogen-free conditions in alaminar air flow unit and supplied with sterile food and water. In thetoxicity studies, 5-7 week old inbred BALB/c mice (Taconic) were used inaddition to the SCID mice.

Establishment and passaging of the xenotransplanted tumors was performedas described (Douglas et al., 1998, Cytometry 32:57-65; Kawamata et al.,1998, Blood 91:561-569; Pepper et al., 1998, Leuk. Res. 22:439-444). Todeplete macrophages and NK cells and to enhance tumor engraftment, SCIDmice were injected intraperitoneally with 30-45 milligrams per kilogramof etoposide (Bedford Laboratories, Bedford, Ohio) 4 days prior toimplantation of the human EBV⁺ B-cell lines (Visonneau et al., 1998, Am.J. Path. 152:1299-1311). Ten million cells of each line employed (seeresults) were inoculated into mice either intraperitoneally orsubcutaneously in 200 microliters of Dulbecco's Phosphate BufferedSaline (DPBS; BioWhittaker, Walkersville, Md.). Ascites or palpablesubcutaneous tumors developed 3-5 weeks after cell injection.

Tumor treatment and growth inhibition experiments were performed usingfragments of the established subcutaneous tumors (Pasqualucci et al.,1995, Blood 85:2139-2146, Visonneau et al., 1998, Am. J. Path.152:1299-1311). For this purpose, mice were anesthetized with KETALAR™(ketamine, Parke-Davis, Morris Plains, N.J.) by intraperitonealinjection of 100 milligrams per kilogram. Next, the primary tumor wasaseptically removed and freed from necrotic, fatty and connective tissueand divided into small pieces of roughly equal size and 3-4 pieces permouse were injected subcutaneously. Treatment of established tumors withSDZ RAD was started when tumors reached 5 millimeters diameter. Ingrowth inhibition experiments, the treatment was initiated 3 days priorto the tumor implantation. Tumor volume in all experiments wasdetermined from the equation: volume=0.4ab², where a and b designaterespectively long and short diameter of the tumor. The transplanted micewere monitored for tumor growth for a period of up to 2 months. Fivemilligrams per kilogram of SDZ RAD was administered once a day by gavageas described (Schuurman et al., 1997, Transplantation 64:32-5; Schuleret al. 1997, Transplantation 64:36-42).

Mice were sacrificed by exposure to FORANE™ (isoflurane, Ohmeda, LibertyPlace, N.J.) on day 29 in the drug toxicity study, on day 40-55 in thetumor growth inhibition study or when tumors achieved approximately 2centimeter diameter, or when ulceration of the skin, signs of severerespiratory distress, weakness, or lethargy appeared. Complete autopsywas performed on all mice at the end of the study regardless of theirappearance. Tumor and internal organs (spleen, liver, lung, heart,kidney, small and large intestines, femoral bone for bone marrow) werefixed in 10% (v/v) formalin, embedded in paraffin, cut into 0.4 micronsections, transferred to glass slides, and stained with hematoxylin andeosin. Representative tumor fragments were stained immunohistochemicallyby standard streptavidin-biotin complex technique using commerciallyavailable reagents (Research Genetics, Huntsville, Ala.), antibodies:anti-CD20 (L-26) and LMP-1 (both from DAKO, Carpinteria, Calif.) andKi67 (mib1; Immunotech, Westbrook, Me.). EBV-encoded RNA (EBER-1), anddetected using commercially available reagents.

The results of the experiments presented in this Example are nowdescribed.

SDZ RAD Inhibits Growth of EBV⁺ B Cells In Vitro

In order to determine whether SDZ RAD can inhibit proliferation of cellsmimicking PTLDs, 6 different EBV⁺ B cell lines were cultured in vitro inthe presence of the drug at various concentrations. Three HTLV-I⁺ T-celllines) resistant to SDZ RAD (Zhang et al., 1999, Leukemia Res.,23:373-384) were used as controls. As shown in FIG. 1, all PTLD-likeEBV⁺ B cell lines were very sensitive to SDZ RAD. A dose of SDZ RAD assmall as 1 nanomolar produced 60-95% inhibition of growth in all celllines. This result was comparable to the inhibition of stimulated normalT lymphocytes (Schuurman et al., 1997, Transplantation 64:32-5; Bohleret al., 1998, Transplant. Proc. 30:2195-2197). Some subtle differencesin the degree of response were noted among the EBV⁺ B-cell lines. Thelines derived from patients with B-cell lymphomas (15A, 20A and BC-1;see Materials and Methods), and thus resembling more the advanced formsof PTLD, tended to show a lower degree of inhibition (80-90%). Celllines obtained from normal B cells (i.e., mimicking the less advancedtypes of PTLD) were inhibited more profoundly (90-100%). The cell lineswere pulsed for 18 hours with tritiated thymidine after 32 hours culturewith 0-10 nanomolar SDZ RAD.

SDZ RAD Blocks Cell Cycle Progression in EBV⁺ B Cells

Rapamycin and, presumably, O-alkylated rapamycin derivatives such as SDZRAD, block cell cycle progression at the early stage in normal T cells(Terada et al., 1993, J. Cell Physiol. 154:7-15; Flanagan et al., 1993,Ann. N.Y. Acad. Sci. 696:31-37). Our cell growth inhibition data whichmeasured incorporation of thymidine (FIG. 1), suggested that SDZ RADinhibits cell cycle progression in PTLD-like B cells. As shown in FIG.2, SDZ RAD markedly inhibited cell cycle progression at the early, G₀/G₁phase in all 4 PTLD-like B cell lines investigated: 20A, A1, A2D6, andLCL. The effect was drug dose and cell-line type dependent. Whereas thelow SDZ RAD doses (1-2 nanomolar) increased the percentage of cells inG₀/G₁ by 5-25, the highest dose tested (10 nanomolar) resulted in the10-70% increase. Percentages of cells in the later phases of the cellcycle (G₂-M and S) were diminished proportionally. A1 and, to a lesserdegree, A2D6 cell line were particularly sensitive to SDZ RAD. Four EBV⁺B-cell lines were cultured for 48 hours with 0-10 nanomolar SDZ RAD,labeled with propidium iodine, and analyzed by flow cytometry.

SDZ RAD Increases Apoptosis in EBV⁺ B Cells

Previous studies have shown that immunosuppressive macrolides asrepresented by rapamycin can induce or enhance apoptosis stimulated byother agents (Gottschalk et al., 1994, Proc. Natl. Acad. Sci. USA91:7350-7354; Muthukkumar et al., 1995, Transplantation 60:264-270; Shiet al., 1995, Cancer Res. 55:1982-1988; Hosoi et al., 1999, Cancer Res.59:886-894). In order to determine if SDZ RAD affects the apoptotic ratein PTLD-like cells, we tested apoptosis-induced surface expression ofAnnexin V by flow cytometry in the 20A, A1, A2D6 cell lines (FIG. 3).Even the lowest SDZ RAD dose (1 nanomolar) markedly increased thepercentage of apoptotic cells in all cell lines, with some differencesresembling the cell growth and cell cycle inhibition data. In the mostsensitive A1 line, SDZ RAD resulted in approximately 60% increase in thenumber of apoptotic cells from 10 to 16%. In A2D6 line, it led to 11%increase, and in the 20A line to 12% increase. At the highest dose (10nanomolar), SDZ RAD led to approximately 240% increase in A1 line and45% increase in both A2D6 and 20A lines.

Toxicity Study

Previous studies have shown that a minimal effective dose of SDZ RAD inrats was 5 milligrams per kilogram body weight per day and >5 milligramsper kilogram body weight per day in the in kidney and heart transplantmodel, respectively, when SDZ RAD was used as a single immunosuppressiveagent (Schuurman et al., 1997, Transplantation 64:32-5; Schuler et al.1997, Transplantation 64: 36-42). In order to determine if prolongedexposure to SDZ RAD is non-toxic for mice, both immunocompetent andimmunodeficient, a cohort of normal BALB/c mice were treated with theabove dose of SDZ RAD for 28 days. Ten treated and 5 control, untreatedmice were used in this study. Later SCID mice which were transplantedwith human PTLD-like lymphomas and treated with SDZ RAD for up to 55days were evaluated. Seven treated and 5 control untreated SCID micewere evaluated in this experiment. After receiving the final dose of thedrug, all mice were sacrificed and the following organs were harvestedfor histopathologic evaluation to investigate for possible toxiceffects: liver, spleen, kidney, small intestine, large intestine, heart,lung, and femoral bone for bone marrow.

None of the treated mice showed any visible signs of drug toxicity.Growth and weight gain were the same for the SDZ RAD-treated and thecontrol group. Microscopic evaluation of the organs of all treated anduntreated mice revealed no pathologic changes which could be attributedto the drug. We conclude, therefore, that prolonged exposure to SDZ RADat the dose of 5 milligrams per kilogram body weight per day has noadverse effect in the treated mice, regardless of their immune status.

Establishment of the xenotransplant model of human PTLD-like lymphoma inSCID mice.

In these experiments, mice were injected with EBV⁺ B-cell lines via twodifferent routes: intraperitoneal and subcutaneous. In theintraperitoneal tumor model, SCID mice (5 per group) were inoculatedintraperitoneally with about 10⁷ cells per line from four cell lines:15A, 20A, A2D6 and BC-1. After 21-35 days, all mice developed fataldisease with ascites and symptoms of weakness or lethargy. Autopsyrevealed extensive tumor infiltrates involving peritoneal wall, liver,spleen, and kidneys. Microscopic examination of the tumors showed alarge cell lymphoma with an infiltrative growth pattern, high mitoticrate, focal and single-cell necrosis and some degree of plasmacytoiddifferentiation. Foci of the lymphoma were also present in distantorgans such as lungs and bone marrow indicating hematogeneous spread.

In the subcutaneous model, mice (also 5 per group) were injected withabout 10⁷ cells from the 15A, 20A and A1 lines. After 21-32 days, allmice developed tumors in the site of implantation. Autopsy revealedsubcutaneous tumors invading adjacent skeletal muscle and skin. Therewas no gross evidence of distant spread of the tumor. Microscopicexamination revealed the same type of high-grade lymphoma as in theintraperitoneal model. Distant internal organs, mainly liver and lungsoccasionally displayed small tumor foci. This occurred only when thesubcutaneous lymphoma reached a relatively large size of at least 1.5 cmin diameter. Immunohistochemical staining confirmed that the tumors werederived from the implanted human EBV⁺ B lymphocytes. The images of the20A line shown in FIG. 4A-D are representative for all three cell lines.

Virtually all lymphoma cells were positive for human B-cell marker CD20.Most (50-80%) were positive for cell-cycle related Ki-67 antigenconsistent with their high proliferative rate. Staining for EBV-relatedantigen EBER1 was universally positive (100% of cells). Twenty to 50% ofcells expressed EBV-associated latent membrane protein 1 (LMP-1). Theseresults confirm that tumors represent human EBV⁺ B-cell lymphomacorresponding to the monomorphic type of PTLD.

The subcutaneous lymphoma model had some advantages over the peritonealmodel. First, even small lymphomas could be easily identified. Second,the lymphomas of up to 1.5 centimeters diameter remained localized whichpermitted determination of total tumor volume with great accuracy (Wasiket al., 1994, Am. J. Path. 144:1089-1097). Finally, the subcutaneoustumor could be transferred simultaneously into several mice byimplanting tumor tissue fragments rather than single-cell suspension(Wasik et al., 1994, Am. J. Path. 144:1089-1097; Pasqualucci et al.,1995, Blood 85:2139-2146; Visonneau et al., 1998, Am. J. Path.152:1299-1311). Such implantation results in a fast establishment oftumors with very similar growth characteristics in virtually allrecipient mice (Wasik et al., 1994, Am. J. Path. 144:1089-1097;Pasqualucci et al., 1995, Blood 85:2139-2146; Visonneau et al., 1998,Am. J. Path. 152:1299-1311). For these reasons, we selected thesubcutaneous model for further studies.

Treatment of Established EBV⁺ B-Cell Tumors.

In order to determine the in vivo effect of SDZ RAD on PTLD-like cells,15A, 20A, and A1 cell line tumors were implanted into 7-15 SCID mice percell line. Treatment was initiated once the tumors reached 5 millimetersdiameter, which corresponds to a volume of 50 cubic millimeters. SDZ RADwas administered by daily gavage at 5 milligrams per kilogram, whichrepresents a non-toxic and effective immunosuppressive dose (Schuurmanet al., 1997, Transplantation 64:32-5; Schuler et al. 1997,Transplantation 64:36-42).

As shown in FIG. 5, SDZ RAD had a profound inhibitory effect on growthof the xenotransplanted PTLD-like tumors with visible differences in thedegree of response among the three tumors. In mice implanted with 15Atumors, there was a marked drug-induced delay of the tumor growth but noabsolute tumor growth inhibition or regression. On day 21, the mediantumor volume was approximately 240 cubic millimeters in the treated miceand 2720 cubic millimeters in the control, untreated mice. As late as onday 38, 15A tumors in the treated mice reached only around 1000 cubicmillimeters. The effect of SDZ RAD on the 20A tumors was even morestriking. Whereas on day 19, the control 20A tumors displayed the medianvolume of 1440 cubic millimeters, the average treated tumor measuredonly 50 cubic millimeters, which was equal to the initial tumor size.Furthermore, a significant regression in the tumor volume was seen in 6out of 10 treated mice. Accordingly, the median tumor volume on day 53was only <5 cubic millimeters for this subset.

SDZ RAD proved to be the most effective against the A1 cell line. On day21 median volume of the treated tumors was 50 cubic millimeters, withnone of the tumors (0/8) showing any evidence of growth. Mean volume ofthe untreated tumors on that day was approximately 1800 cubicmillimeters. Further treatment resulted in a steady regression in all 8mice. On day 53, the mean tumor volume decreased to <5 cubicmillimeters, and no lymphoma could be detected in 4 mice, indicatingtotal tumor eradication. The other 4 mice had microscopically-confirmedresidual lymphoma. Noteworthy, the differences in the in vivoeffectiveness of SDZ RAD against the 15A, 20A, and A1 tumors paralleledthe differences seen in the proliferation (FIG. 1) and the other invitro assays (FIGS. 2 and 3). This observation suggests that the cellanalysis in vitro is predictive of the response to SDZ RAD in vivo.

Inhibition of Tumor Establishment

Because SDZ RAD, when used as an immunosuppressive agent, isadministered chronically to transplant patients, its main therapeuticimpact on PTLDs may be to inhibit their development rather than toalleviate the clinically symptomatic cases. To test if SDZ RAD caninhibit establishment of PTLD-like lymphomas, daily treatment with 5milligrams per kilogram body weight of the drug was initiated 3 daysprior to the tumor implantation. As shown in FIG. 6, SDZ RAD proved tobe extremely effective in this model. In 15A tumors, which are the leastsensitive to SDZ RAD (FIG. 5), treatment with SDZ RAD profoundly delayedthe tumor growth, but was unable to inhibit the tumor establishment. Onday 25, the treated tumors measured in average 38 cubic millimeters, theuntreated approximately 1940 cubic millimeters. On day 45, the medianvolume of the treated tumor was 480 cubic millimeters. In 20A tumors, 6out of 11 tumors were not detectable on day 25; the median volume of theremaining 5 was 20 cubic millimeters. The average untreated tumormeasured 210 cubic millimeters on that day. On day 45, none of thetreated tumors exceeded 150 cubic millimeters, the average untreatedtumor measured approximately 4200 cubic millimeters. Five treated miceshowed no signs of tumor; microscopic evaluation of the implantationsite revealed no malignant cells. Finally, in A1 mice no tumor could bedetected as late as on day 29 in any of the 8 treated mice; the averagetumor in the untreated mice measured 580 cubic millimeters. On day 53only 3 mice showed small (<5 cubic millimeters), histologicallyconfirmed lymphomas. The remaining 5 mice showed no evidence of tumor.

Whereas the standard immunosuppressive agents foster development ofPTLDs, the impact of SDZ RAD and other macrolides on these disordersremains undetermined. It was observed that SDZ RAD profoundly suppressedin vitro lymphocyte proliferation, blocked cell cycle progression, andincreased apoptotic rate in PTLD-like EBV⁺ B cells. In the in vivo SCIDmouse xenotransplant model, SDZ RAD markedly delayed growth or inducedregression of established EBV⁺ B-cell tumors. The drug completelyeradicated or inhibited tumor establishment in a subset of the treatedmice. These findings indicate that macrolides such as SDZ RAD may beeffective in inhibition and treatment of PTLDs.

SDZ RAD profoundly suppressed in vitro proliferation, arrested cellcycle progression at the early, G₀/G₁ stage, and increased apoptoticrate in PTLD-like EBV⁺ B cells. In the SCID mice xenotransplanted withthree different PTLD-like B-cell tumors, SDZ RAD markedly inhibitedgrowth of these tumors, particularly if administered prior to tumorimplantation. In the A1 tumors, total eradication of already establishedtumors was achieved in 4 out of 8 (4/8) mice. In two of the three linestested (20A and A1), SDZ RAD completely inhibited establishment oftumors in approximately 50% of mice (5/11 and 5/8, respectively). Theanti-EBV⁺ B-cell tumor property of SDZ RAD contrasts with the standardimmunosuppressive agents cyclosporin and tacrolimus, which have beensuggested recently to enhance outgrowth of EBV⁺ B cells, not onlyindirectly by suppressing an immune response against such cells, butalso directly by protecting them from the effects of pro-apoptoticsignals (Beatty et al., 1998, Transplantation 65:1248-1255.)

The results of these experiments are particularly encouraging because15A and 20A lines were derived from lymphoma cells (Silberstein et al.,1991, Blood 78:2372-2386) and, as such, appear to correspond to theadvanced, clinically aggressive form of PTLD. It is plausible,therefore, that other, less malignant forms of PTLD which comprisemajority of cases, should be even more sensitive to SDZ RAD. The factthat the A1 cell line obtained from a normal individual was moresensitive than 15A and 20A to SDZ RAD, supports this assumption.

The data presented in this Example also suggest that monotherapy withSDZ RAD may not be sufficient to eradicate some established, overtlymalignant PTLD tumors, and that combination therapy using both SDZ RAD(or another O-alkylated rapamycin derivative or rapamycin) and one ormore conventional chemotherapeutic drugs can considered in the clinicalsetting (Shi et al., 1995, Res. 55:1982-1988; Mild et al., 1998,Transplant. Proc. 30:1091-1092).

Although inhibition of cell growth, as measured by thymidineincorporation (FIG. 1) and cell cycle progression (FIG. 2), appears tobe the main mode of SDZ RAD action on EBV⁺ B cells, the resultsdescribed herein that SDZ RAD also increases apoptotic rate in suchcells (FIG. 3), suggests that programmed cell death may also be animportant component of anti-tumor activity of the drug. Thispro-apoptotic effect can be particularly important in treatment ofestablished PTLD tumors, where inhibition of tumor growth alone mightnot be sufficient to achieve complete tumor regression. The observationherein that only prolonged exposure to SDZ RAD led to marked regressionor elimination of many tumors (FIG. 5), suggests that a similar extendedtreatment may be desirable to eradicate established PTLDs.

Although PTLD-like B-cell lines sensitive to SDZ RAD were all EBV⁺, thepotential role, if any, of the virus in mediating this sensitivity isnot fully understood. EBV encodes or induces in the target cells severalproteins capable of activating cytokine signaling pathways (Rochford, etal., 1997, Viral Immunol 10:183-195). The membrane-anchored viralprotein designated LMP-1 is the best characterized, and uses the TRAFsignaling pathway of the TNF receptor family (Mosialos et al., 1995,Cell 80:389-399; Devergne et al., 1998, J. Virol. 72:7900-7908;Liebowitz et al., 1998, New Eng. J. Med. 338:1413-1421). It isinteresting in this context, that signaling via CD40 which belongs tothe family and shares several features with LMP-1 including signalingvia TRAF3 protein (Eliopoulos et al., 1996, Oncogene 13:2243-2254;Pullen et al., 1998, Biochemistry 37:11836-11845) has been shownrecently to be inhibited by rapamycin (Sakata et al., 1999, ImmunologyLetters 68:301-309). Immunohistochemical analysis indicates that LMP-1is expressed only by a subset of EBV⁺ B cells, which suggests that LMP-1may not be critical for growth of the PTLD-like cells and theirsensitivity to SDZ RAD. EBV encodes also two other, related membraneproteins, designated LMP-2A and LMP-2B. These proteins do not appear tobe essential for in vivo growth of EBV⁺ B cells (Rochford et al., 1997,Arch Virol 142:707-720). Alternatively, SDZ RAD might inhibit signalingmediated by cytokines induced by EBV in the target cells such asTNF-alpha and TNF-beta (Rochford, et al., 1997, Viral Immunol10:183-195).

SDZ RAD exhibits potent inhibitory activity on the PTLD-like, EBV⁺lymphoblastoid B-cell lines, both in vitro and in vivo. SDZ RADprofoundly inhibits in vitro proliferation of such cells and arrestedtheir cell cycle progression at the early, G₀/G₁ stage. In addition, thedrug increases apoptotic rate of EBV⁺ B cells. In vivo, it markedlydelays or completely inhibits growth of EBV⁺ B cells xenotransplantedinto SCID mice, particularly when administered prior to cellimplantation. SDZ RAD is able to eradicate an established tumor in someinstances. This effect appears to be cell line-specific and proportionalto the drug effect seen in vitro.

In summary, the data presented in this Example demonstrate that SDZ RADhas a potent inhibitory effect on EBV⁺ B lymphocytes in vitro and invivo. Therefore, it can be effective in treatment and inhibition ofPTLDs in transplant patients or in treatment and inhibition of otherlymphoproliferative disorders.

New treatment modalities are needed to inhibit development and improvecure rate of PTLDs. An ideal anti-PTLD drug would play a double role ofinhibiting graft rejection and, at the same time, inhibiting developmentand growth of PTLD. Should PTLD develop despite the treatment, anincrease, rather than a decrease in the drug dose as is currently donewith the standard immunosuppressive drugs, might be effective. Thisapproach of increasing the drug dose would have an additional advantageof not jeopardizing survival of the graft. This Example presents invitro and in vivo data which indicate that SDZ RAD, a macrolideimmunosuppressive agent, has the dual roles of an anti-rejection agentand an anti-lymphoproliferative agent.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention can be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims include all such embodiments and equivalent variations.

1-29. (canceled)
 30. A method of alleviating a lymphoproliferativedisorder in a human patient, the method comprising administering to thepatient, in an amount sufficient to alleviate the disorder, a rapamycinderivative having the chemical structure

wherein X is (H, H) or O; Y is (H, OH) or O; R¹ is selected from thegroup consisting of alkyl, thioalkyl, arylalkyl, hydroxyalkyl,dihydroxyalkyl, hydroxyalkylarylalkyl, dihydroxyalkylarylalkyl,alkoxyalkyl, acyloxyalkyl, aminoalkyl, alkylaminoalkyl,alkoxycarbonylaminoalkyl, acylaminoalkyl, arylsulfonamidoalkyl, allyl,dihydroxyalkylallyl, dioxolanylallyl, carbalkoxyalkyl, and (R³)₃ Si; R²is selected from the group consisting of —H, alkyl, thioalkyl,arylalkyl, hydroxyalkyl, dihydroxyalkyl, hydroxyalkylarylalkyl,dihydroxyalkylarylalkyl, alkoxyalkyl, acyloxyalkyl, aminoalkyl,alkylaminoalkyl, alkoxycarbonylaminoalkyl, acylaminoalkyl,arylsulfonamidoalkyl, allyl, dihydroxyalkylallyl, dioxolanylallyl,carbalkoxyalkyl, and (R³)₃ Si; each R³ is independently selected fromthe group consisting of —H, methyl, ethyl, isopropyl, tert-butyl, andphenyl; and either R⁴ is methyl or R⁴ and R¹ together form a C₂₋₅alkylene moiety, wherein the lymphoproliferative disorder is a B-celllymphoma.
 31. The method of claim 30, wherein the rapamycin derivativeis selected from the group consisting of 40-O-benzyl-rapamycin,40-O-(4′-hydroxymethyl)benzyl-rapamycin, 40-O-[4′-(1,2dihydroxyethyl)]benzyl-rapamycin, 40-O-Allyl-rapamycin,40-O-[3′-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2′-en-1′-yl]-rapamycin,(2′E, 4′S)-40-O-(4′,5′-dihydroxypent-2′-en-1′-yl)-rapamycin,40-O-(2-hydroxy)ethoxycarbonylmethyl-rapamycin,40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin,40-O-(6-hydroxy)hexyl-rapamycin,40-O-[2-(2hydroxy)ethoxy]ethyl-rapamycin,40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin,40-O-[(2S)-2,3-dihydroxyprop-1-yl]rapamycin,40-O-(2-acetoxy)ethyl-rapamycin, 40-O-(2-nicotinoyloxy)ethyl-rapamycin,40-O-[2-(N-morpholino)acetoxy]ethyl-rapamycin,40-O-(2-N-imidazolylacetoxy)ethyl-rapamycin,40-O-[2-(N-methyl-M-piperazinyl)acetoxy]ethyl-rapamycin,39-O-desmethyl-39,40-O,O-ethylene-rapamycin,(26R)-26-dihydro-40-O-(2-hydroxy)ethyl-rapamycin, 28-O-methyl-rapamycin,40-O-(2-aminoethyl)-rapamycin, 40-O-(2-acetaminoethyl)-rapamycin,40-O-(2-nicotinamidoethyl)-rapamycin,40-O-(2-(N-methyl-imidazo-2′-ylcarbethoxamido)ethyl)-rapamycin,40-O-(2-ethoxycarbonylaminoethyl)-rapamycin,40-O-(2-tolylsulfonamidoethyl)-rapamycin, and40-O-[2-(4′5′-dicarboethoxy-1′,2′,3′-triazol-1′-yl)-ethyl]-rapamycin.32. The method of claim 30, wherein the rapamycin derivative is selectedfrom the group consisting of 40-O-(2-hydroxy)ethyl-rapamycin,40-O-(3-hydroxy)propyl-rapamycin,40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, and40-O-(2-acetaminoethyl)-rapamycin.
 33. The method of claim 30, whereinthe rapamycin derivative is 40-O-(2-hydroxy)ethyl-rapamycin.
 34. Themethod of claim 30, wherein the rapamycin derivative is co-administeredwith a second pharmacologically active agent.
 35. The method of claim34, wherein the second agent is selected from the group consisting ofcisplatin and a cyclophosphamide.
 36. The method of claim 30, whereinthe rapamycin derivative and the second agent are administered to thepatient in a composition comprising both the rapamycin derivative andthe second agent.
 37. The method of claim 30, wherein the rapamycinderivative is administered to the patient at least one hour before thesecond agent is administered to the patient.
 38. The method of claim 30,wherein the rapamycin derivative is administered to the patient at leastone hour after the second agent is administered to the patient.
 39. Themethod of claim 30, wherein the B-cell lymphoma is selected from thegroup consisting of an AIDS-related lymphoma, a Hodgkin's lymphoma, anon-Hodgkin's lymphoma, a Burkitt's lymphoma, a monoclonal overtlymalignant B-cell lymphoma, a diffuse large cell lymphoma, and aperitoneal effusion B-cell lymphoma.
 40. The method of claim 30, whereinthe amount is from 10 micrograms per kilogram body weight to 5milligrams per kilogram body weight.
 41. The method of claim 30, whereinthe rapamycin derivative is administered parenterally to the patient.